Powassan Virus Lineage I in Field-Collected Dermacentor variabilis Ticks, New York, USA

Powassan virus is a tickborne flavivirus that can cause lethal or debilitating neurologic illness. It is canonically transmitted by Ixodes spp. ticks but might spill over to sympatric Dermacentor species. We detected Powassan virus lineage I from a pool of field-collected D. variabilis ticks in New York, USA.

spp. ticks have been of particular interest because of their common occurrence in POWV-and DTVendemic areas and because of their tendency to bite humans. POWV has been isolated from D. andersoni ticks in Colorado, USA (6); genetic analysis suggests that strain, called POWV 791A-52, is most likely a form of DTV (7). However, it remains unclear whether the tick in question was infected by spillover from another sylvatic cycle featuring Ixodes ticks or constitutes its own sylvatic system. Neither I. scapularis nor I. cookei ticks are native to Colorado, although several rodent-specific Ixodes species are present (8) that might be involved in such a system.
The competency of D. andersoni ticks for POWV has been confirmed under laboratory conditions when fed from artificially inoculated nonnative species (9). Recent analysis has also indicated that D. variabilis ticks are capable of acquiring and transmitting DTV under laboratory conditions, including maintaining replicating virus transstadially (10). Although that capability has been confirmed in experimentally infected ticks, it remains unclear whether wild populations of D. variabilis ticks can maintain and transmit POWV or DTV under natural circumstances. Considering that D. variabilis ticks are the second most common human-biting species in New York, USA, (11), the ability for the species to transmit POWV in nature represents a critical component of potential human exposure. We detected POWV lineage I from D. variabilis ticks collected in New York in 2021.

The Study
As a part of ongoing efforts to track the emergence of POWV in New York, we performed tick surveillance in areas known to contain circulating POWV as identified from a community-engaged tick testing program (11). From 1 area of interest in Dutchess County, New York, we collected 5 female and 3 male D. variabilis ticks, in addition to 68 I. scapularis ticks, in the second half of April 2021. We visually speciated the ticks and assessed them for feeding status. The female D. variabilis ticks were unfed; we pooled, homogenized, and tested them for POWV by quantitative reverse transcription PCR as described (11). In brief, we initially detected POWV with a primer sensitive to both POWV lineage I and DTV. Then, we used a differentiation quantitative reverse transcription PCR to confirm POWV lineage I with a titer of 3.88 log 10 FFU/ μg RNA. In contrast, none of the I. scapularis ticks collected from the same site tested positive for POWV lineage I and DTV. We used our highly multiplexed PCR amplicon approach to sequence POWV detected from the tick homogenate (12). We prepared libraries with the Illumina COVIDSeq Test (RUO version; Illumina, https://www.illumina.com), replacing the SARS-CoV-2 primers with POWV (13), and sequenced on the Illumina NovaSeq at the Yale Center for Genome Analysis (New Haven, CT, USA). Consensus genomes were generated at a minimum nucleotide frequency threshold of 0.75 and minimum depth of 10 reads using iVar version 1.3.1 (https://github.com/ andersen-lab/ivar).
We reconstructed a maximum-likelihood phylogenetic tree of 29 aligned POWV genomes trimmed to the coding sequence (genome positions 108-10,352) (Figure) using IQ-TREE version 1.6.12 (http://www. iqtree.org) with ultrafast bootstrap approximation (1,000 replicates) (14). Our phylogenetic analysis revealed that the virus (deposited in GenBank under accession no. OM681505) belongs to the POWV lineage I clade and is closely related to a POWV lineage I virus that we sequenced from I. cookei ticks (GenBank accession no. OM681504) from New York in 2020 (Figure). In addition, we used Sanger sequencing of tick ribosomal RNA to confirm that the sample was derived from D. variabilis ticks and not another potential vector species. The resulting sequence (GenBank accession no. ON922563) had 100% homology to D. variabilis large subunit ribosomal RNA (GenBank accession no. L34300.1).
Our results confirm POWV in D. variabilis ticks in southern New York, suggesting that POWV can exist in this tick species, either because of incidental exposure or because of its own sylvatic cycle. In this study, the POWV we identified groups with lineage I; this 416 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 2, February 2023 lineage is normally associated with I. cookei ticks and woodchucks (Marmota monax), instead of with I. scapularis ticks and Peromyscus leucopus mice (15). This link suggests either spillover from that sylvatic cycle, a unique D. variabilis species-dependent sylvatic cycle for POWV lineage I, or a unique subtype of the virus specific to D. variabilis ticks with an unknown sylvatic cycle. Regardless of its source, our data indicate that some D. variabilis ticks in POWV-endemic areas could be capable of acquiring a genotype of POWV similar to lineage I.

Conclusions
POWV is a medically noteworthy flavivirus understood to be primarily transmitted by Ixodes spp. ticks in North America. The sympatric tick species D. variabilis, however, has been recently demonstrated to be a competent vector for POWV under laboratory conditions (10). We report the detection of POWV lineage I in D. variabilis ticks collected from the wild, suggesting that the species might play a direct role in POWV transmission in nature. The ability of POWV to infect humans and the nature of the disease it causes remain unclear, and further research is needed to understand the role of D. variabilis ticks in the ecology of POWV. However, considering that D. variabilis ticks are a primary species of human-biting ticks in New York, this finding demonstrates a new potential source of human exposure to POWV.
The study described in this manuscript was funded by Departmental Start-up funds, Upstate Foundation (Fund ID: 23709) and SUNY Empire Innovation Professorship funds to S.T. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

About the Author
Dr. Hart is a postdoctoral researcher at Upstate Medical University in Syracuse, New York, USA. His primary research focus is on the interaction between vector-borne pathogens, arthropod vectors, and vector-host interactions.